Polypeptides having aspartic endopeptidase activity and polynucleotides encoding same

ABSTRACT

The present invention relates to isolated polypeptides having aspartic endopeptidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofPCT/US2009/067562 filed on Dec. 10, 2009 and claims priority from U.S.provisional application Ser. No. 61/122,173 filed on Dec. 12, 2008,which applications are fully incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing filed electronically byEFS, which is incorporated herein by reference.

REFERENCE TO A DEPOSIT OF BIOLOGICAL MATERIAL

This application contains a reference to a deposit of biologicalmaterial, which deposit is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to isolated polypeptides having asparticendopeptidase activity and isolated polynucleotides encoding thepolypeptides. The invention also relates to nucleic acid constructs,vectors, and host cells comprising the polynucleotides as well asmethods of producing and using the polypeptides.

2. Description of the Related Art

Aspartic endopeptidases are a family of proteolytic enzymes found in adiverse range of species. The best known sources of asparticendopeptidase are the stomach of mammals, yeast, and fungi. Theseenzymes are all characterized by the presence of two aspartic acidresidues at the active site. Specificity is typically for residues withlarge hydrophobic sidechains on either side of the scissile bond.

Aspartic endopeptidases are known to be produced by several fungi suchas Mucor miehei, Rhizomucor pusillus, and Aspergillus species (J.Polaina and A. P. MacCabe, eds., Industrial Enzymes, 207-219; Majima etal., 1988, Agric. Biol. Chem. 52: 787-793). However, there is a need inthe art for alternative sources of aspartic endopeptidases.

The present invention provides polypeptides having asparticendopeptidase activity and polynucleotides encoding the polypeptides.

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides having asparticendopeptidase activity selected from the group consisting of:

(a) a polypeptide comprising an amino acid sequence having at least 60%sequence identity to the mature polypeptide of SEQ ID NO: 2;

(b) a polypeptide encoded by a polynucleotide that hybridizes under atleast medium stringency conditions with (i) the mature polypeptidecoding sequence of SEQ ID NO: 1, (ii) the genomic DNA sequencecomprising the mature polypeptide coding sequence of SEQ ID NO: 1, or(iii) a full-length complementary strand of (i) or (ii);

(c) a polypeptide encoded by a polynucleotide comprising a nucleotidesequence having at least 60% sequence identity to the mature polypeptidecoding sequence of SEQ ID NO: 1; and

(d) a variant comprising a substitution, deletion, and/or insertion ofone or more (several) amino acids of the mature polypeptide of SEQ IDNO: 2.

The present invention also relates to isolated polynucleotides encodingpolypeptides having aspartic endopeptidase activity, selected from thegroup consisting of:

(a) a polynucleotide encoding a polypeptide comprising an amino acidsequence having at least 60% sequence identity to the mature polypeptideof SEQ ID NO: 2;

(b) a polynucleotide that hybridizes under at least medium stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the genomic DNA sequence comprising the mature polypeptidecoding sequence of SEQ ID NO: 1, or (iii) a full-length complementarystrand of (i) or (ii);

(c) a polynucleotide comprising a nucleotide sequence having at least60% sequence identity to the mature polypeptide coding sequence of SEQID NO: 1; and

(d) a polynucleotide encoding a variant comprising a substitution,deletion, and/or insertion of one or more (several) amino acids of themature polypeptide of SEQ ID NO: 2.

The present invention also relates to nucleic acid constructs,recombinant expression vectors, and recombinant host cells comprisingthe polynucleotides, and to methods of producing the polypeptides havingaspartic endopeptidase activity.

The present invention also relates to methods of inhibiting theexpression of a polypeptide having aspartic endopeptidase activity in acell, comprising administering to the cell or expressing in the cell adouble-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises asubsequence of a polynucleotide of the present invention. The presentalso relates to such a double-stranded inhibitory RNA (dsRNA) molecule,wherein optionally the dsRNA is a siRNA or a miRNA molecule.

The present invention also relates to methods of using a polypeptidehaving aspartic endopeptidase activity.

The present invention also relates to plants comprising an isolatedpolynucleotide encoding a polypeptide having aspartic endopeptidaseactivity.

The present invention also relates to methods of producing a polypeptidehaving aspartic endopeptidase activity, comprising: (a) cultivating atransgenic plant or a plant cell comprising a polynucleotide encodingthe polypeptide having aspartic endopeptidase activity under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide.

The present invention also relates to an isolated polynucleotideencoding a signal peptide comprising or consisting of amino acids 1 to20 of SEQ ID NO: 2; to nucleic acid constructs, expression vectors, andrecombinant host cells comprising the polynucleotide; and to methods ofproducing a protein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the cDNA sequence and the deduced amino acid sequence of aThielavia terrestris NRRL 8126 aspartic endopeptidase gene (SEQ ID NOs:1 and 2, respectively).

FIG. 2 shows a map of pTter30A5.

DEFINITIONS

Aspartic endopeptidase activity: The term “aspartic endopeptidaseactivity” is defined herein as a proteolytic activity (EC 3.4.23) thatcatalyzes the hydrolysis of peptide bonds in proteins. For purposes ofthe present invention, aspartic endopeptidase activity is determinedaccording to the procedure described by Aikawa et al., 2001, J. Biochem.129: 791-794.

The polypeptides of the present invention have at least 20%, preferablyat least 40%, more preferably at least 50%, more preferably at least60%, more preferably at least 70%, more preferably at least 80%, evenmore preferably at least 90%, most preferably at least 95%, and evenmost preferably at least 100% of the aspartic endopeptidase activity ofthe mature polypeptide of SEQ ID NO: 2.

Isolated polypeptide: The term “isolated polypeptide” as used hereinrefers to a polypeptide that is isolated from a source. In a preferredaspect, the polypeptide is at least 1% pure, preferably at least 5%pure, more preferably at least 10% pure, more preferably at least 20%pure, more preferably at least 40% pure, more preferably at least 60%pure, even more preferably at least 80% pure, and most preferably atleast 90% pure, as determined by SDS-PAGE.

Substantially pure polypeptide: The term “substantially purepolypeptide” denotes herein a polypeptide preparation that contains atmost 10%, preferably at most 8%, more preferably at most 6%, morepreferably at most 5%, more preferably at most 4%, more preferably atmost 3%, even more preferably at most 2%, most preferably at most 1%,and even most preferably at most 0.5% by weight of other polypeptidematerial with which it is natively or recombinantly associated. It is,therefore, preferred that the substantially pure polypeptide is at least92% pure, preferably at least 94% pure, more preferably at least 95%pure, more preferably at least 96% pure, more preferably at least 97%pure, more preferably at least 98% pure, even more preferably at least99% pure, most preferably at least 99.5% pure, and even most preferably100% pure by weight of the total polypeptide material present in thepreparation. The polypeptides of the present invention are preferably ina substantially pure form, i.e., that the polypeptide preparation isessentially free of other polypeptide material with which it is nativelyor recombinantly associated. This can be accomplished, for example, bypreparing the polypeptide by well-known recombinant methods or byclassical purification methods.

Mature polypeptide: The term “mature polypeptide” is defined herein as apolypeptide in its final form following translation and anypost-translational modifications, such as N-terminal processing,C-terminal truncation, glycosylation, phosphorylation, etc. In oneaspect, the mature polypeptide is amino acids 21 to 467 of SEQ ID NO: 2based on the SignalP program (Nielsen et al., 1997, Protein Engineering10:1-6) that predicts amino acids 1 to 20 of SEQ ID NO: 2 are a signalpeptide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” is defined herein as a nucleotide sequence that encodes amature polypeptide having aspartic endopeptidase activity. In oneaspect, the mature polypeptide coding sequence is nucleotides 61 to 1401of SEQ ID NO: 1 based on the SignalP program (Nielsen et al., 1997,supra) that predicts nucleotides 1 to 60 of SEQ ID NO: 1 encode a signalpeptide.

Sequence Identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the degree of sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends in Genetics 16: 276-277), preferably version 3.0.0 orlater. The optional parameters used are gap open penalty of 10, gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the—nobrief option) is used as the percent identity andis calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of sequence identitybetween two deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra), preferably version 3.0.0 or later. The optional parameters usedare gap open penalty of 10, gap extension penalty of 0.5, and theEDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the—nobrief option)is used as the percent identity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Homologous sequence: The term “homologous sequence” is defined herein asa predicted protein having an E value (or expectancy score) of less than0.001 in a tfasty search (Pearson, W. R., 1999, in BioinformaticsMethods and Protocols, S. Misener and S. A. Krawetz, ed., pp. 185-219)with the Thielavia terrrestris polypeptide having aspartic endopeptidaseactivity of SEQ ID NO: 2 or the mature polypeptide thereof.

Polypeptide fragment: The term “polypeptide fragment” is defined hereinas a polypeptide having one or more (several) amino acids deleted fromthe amino and/or carboxyl terminus of the mature polypeptide of SEQ IDNO: 2; or a homologous sequence thereof; wherein the fragment hasaspartic endopeptidase activity. In one aspect, a fragment contains atleast 380 amino acid residues, more preferably at least 400 amino acidresidues, and most preferably at least 420 amino acid residues of themature polypeptide of SEQ ID NO: 2 or a homologous sequence thereof.

Subsequence: The term “subsequence” is defined herein as a nucleotidesequence having one or more (several) nucleotides deleted from the 5′and/or 3′ end of the mature polypeptide coding sequence of SEQ ID NO: 1;or a homologous sequence thereof; wherein the subsequence encodes apolypeptide fragment having aspartic endopeptidase activity. In oneaspect, a subsequence contains at least 1140 nucleotides, morepreferably at least 1200 nucleotides, and most preferably at least 1260nucleotides of the mature polypeptide coding sequence of SEQ ID NO: 1 ora homologous sequence thereof.

Allelic variant: The term “allelic variant” denotes herein any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Isolated polynucleotide: The term “isolated polynucleotide” as usedherein refers to a polynucleotide that is isolated from a source. In apreferred aspect, the polynucleotide is at least 1% pure, preferably atleast 5% pure, more preferably at least 10% pure, more preferably atleast 20% pure, more preferably at least 40% pure, more preferably atleast 60% pure, even more preferably at least 80% pure, and mostpreferably at least 90% pure, as determined by agarose electrophoresis.

Substantially pure polynucleotide: The term “substantially purepolynucleotide” as used herein refers to a polynucleotide preparationfree of other extraneous or unwanted nucleotides and in a form suitablefor use within genetically engineered protein production systems. Thus,a substantially pure polynucleotide contains at most 10%, preferably atmost 8%, more preferably at most 6%, more preferably at most 5%, morepreferably at most 4%, more preferably at most 3%, even more preferablyat most 2%, most preferably at most 1%, and even most preferably at most0.5% by weight of other polynucleotide material with which it isnatively or recombinantly associated. A substantially purepolynucleotide may, however, include naturally occurring 5′ and 3′untranslated regions, such as promoters and terminators. It is preferredthat the substantially pure polynucleotide is at least 90% pure,preferably at least 92% pure, more preferably at least 94% pure, morepreferably at least 95% pure, more preferably at least 96% pure, morepreferably at least 97% pure, even more preferably at least 98% pure,most preferably at least 99% pure, and even most preferably at least99.5% pure by weight. The polynucleotides of the present invention arepreferably in a substantially pure form, i.e., that the polynucleotidepreparation is essentially free of other polynucleotide material withwhich it is natively or recombinantly associated. The polynucleotidesmay be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or anycombinations thereof.

Coding sequence: When used herein the term “coding sequence” means anucleotide sequence, which directly specifies the amino acid sequence ofits protein product. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon or alternative start codons such as GTG and TTG and endswith a stop codon such as TAA, TAG, and TGA. The coding sequence may bea DNA, cDNA, synthetic, or recombinant nucleotide sequence.

cDNA: The term “cDNA” is defined herein as a DNA molecule that can beprepared by reverse transcription from a mature, spliced, mRNA moleculeobtained from a eukaryotic cell. cDNA lacks intron sequences that may bepresent in the corresponding genomic DNA. The initial, primary RNAtranscript is a precursor to mRNA that is processed through a series ofsteps before appearing as mature spliced mRNA. These steps include theremoval of intron sequences by a process called splicing. cDNA derivedfrom mRNA lacks, therefore, any intron sequences.

Nucleic acid construct: The term “nucleic acid construct” as used hereinrefers to a nucleic acid molecule, either single- or double-stranded,which is isolated from a naturally occurring gene or which is modifiedto contain segments of nucleic acids in a manner that would nototherwise exist in nature or which is synthetic. The term nucleic acidconstruct is synonymous with the term “expression cassette” when thenucleic acid construct contains the control sequences required forexpression of a coding sequence of the present invention.

Control sequences: The term “control sequences” is defined herein toinclude all components necessary for the expression of a polynucleotideencoding a polypeptide of the present invention. Each control sequencemay be native or foreign to the nucleotide sequence encoding thepolypeptide or native or foreign to each other. Such control sequencesinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, promoter, signal peptide sequence, andtranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Thecontrol sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleotide sequenceencoding a polypeptide.

Operably linked: The term “operably linked” denotes herein aconfiguration in which a control sequence is placed at an appropriateposition relative to the coding sequence of a polynucleotide sequencesuch that the control sequence directs the expression of the codingsequence of a polypeptide.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” is defined herein as alinear or circular DNA molecule that comprises a polynucleotide encodinga polypeptide of the present invention and is operably linked toadditional nucleotides that provide for its expression.

Host cell: The term “host cell”, as used herein, includes any cell typethat is susceptible to transformation, transfection, transduction, andthe like with a nucleic acid construct or expression vector comprising apolynucleotide of the present invention.

Modification: The term “modification” means herein any chemicalmodification of the polypeptide comprising or consisting of the maturepolypeptide of SEQ ID NO: 2; or a homologous sequence thereof; as wellas genetic manipulation of the DNA encoding such a polypeptide. Themodification can be a substitution, a deletion and/or an insertion ofone or more (several) amino acids as well as replacements of one or more(several) amino acid side chains.

Artificial variant: When used herein, the term “artificial variant”means a polypeptide having aspartic endopeptidase activity produced byan organism expressing a modified polynucleotide sequence of the maturepolypeptide coding sequence of SEQ ID NO: 1; or a homologous sequencethereof. The modified nucleotide sequence is obtained through humanintervention by modification of the polynucleotide sequence disclosed inSEQ ID NO: 1; or a homologous sequence thereof.

DETAILED DESCRIPTION OF THE INVENTION Polypeptides Having AsparticEndopeptidase Activity

In a first aspect, the present invention relates to isolatedpolypeptides comprising amino acid sequences having a degree of sequenceidentity to the mature polypeptide of SEQ ID NO: 2 of preferably atleast 60%, more preferably at least 65%, more preferably at least 70%,more preferably at least 75%, more preferably at least 80%, morepreferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which have aspartic endopeptidaseactivity (hereinafter “homologous polypeptides”). In a preferred aspect,the homologous polypeptides comprise amino acid sequences that differ byten amino acids, preferably by five amino acids, more preferably by fouramino acids, even more preferably by three amino acids, most preferablyby two amino acids, and even most preferably by one amino acid from themature polypeptide of SEQ ID NO: 2.

A polypeptide of the present invention preferably comprises the aminoacid sequence of SEQ ID NO: 2 or an allelic variant thereof; or afragment thereof having aspartic endopeptidase activity. In a preferredaspect, the polypeptide comprises the amino acid sequence of SEQ ID NO:2. In another preferred aspect, the polypeptide comprises the maturepolypeptide of SEQ ID NO: 2. In another preferred aspect, thepolypeptide comprises amino acids 21 to 467 of SEQ ID NO: 2, or anallelic variant thereof; or a fragment thereof having asparticendopeptidase activity. In another preferred aspect, the polypeptidecomprises amino acids 21 to 467 of SEQ ID NO: 2. In another preferredaspect, the polypeptide consists of the amino acid sequence of SEQ IDNO: 2 or an allelic variant thereof; or a fragment thereof havingaspartic endopeptidase activity. In another preferred aspect, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 2. Inanother preferred aspect, the polypeptide consists of the maturepolypeptide of SEQ ID NO: 2. In another preferred aspect, thepolypeptide consists of amino acids 21 to 467 of SEQ ID NO: 2 or anallelic variant thereof; or a fragment thereof having asparticendopeptidase activity. In another preferred aspect, the polypeptideconsists of amino acids 21 to 467 of SEQ ID NO: 2.

In a second aspect, the present invention relates to isolatedpolypeptides having aspartic endopeptidase activity that are encoded bypolynucleotides that hybridize under preferably very low stringencyconditions, more preferably low stringency conditions, more preferablymedium stringency conditions, more preferably medium-high stringencyconditions, even more preferably high stringency conditions, and mostpreferably very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 1, (ii) the genomic DNAsequence comprising the mature polypeptide coding sequence of SEQ ID NO:1, or (iii) a full-length complementary strand of (i) or (ii) (J.Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning, ALaboratory Manual, 2d edition, Cold Spring Harbor, N.Y.).

The nucleotide sequence of SEQ ID NO: 1; or a subsequence thereof; aswell as the amino acid sequence of SEQ ID NO: 2; or a fragment thereof;may be used to design nucleic acid probes to identify and clone DNAencoding polypeptides having aspartic endopeptidase activity fromstrains of different genera or species according to methods well knownin the art. In particular, such probes can be used for hybridizationwith the genomic or cDNA of the genus or species of interest, followingstandard Southern blotting procedures, in order to identify and isolatethe corresponding gene therein. Such probes can be considerably shorterthan the entire sequence, but should be at least 14, preferably at least25, more preferably at least 35, and most preferably at least 70nucleotides in length. It is, however, preferred that the nucleic acidprobe is at least 100 nucleotides in length. For example, the nucleicacid probe may be at least 200 nucleotides, preferably at least 300nucleotides, more preferably at least 400 nucleotides, or mostpreferably at least 500 nucleotides in length. Even longer probes may beused, e.g., nucleic acid probes that are preferably at least 600nucleotides, more preferably at least 700 nucleotides, even morepreferably at least 800 nucleotides, or most preferably at least 900nucleotides in length. Both DNA and RNA probes can be used. The probesare typically labeled for detecting the corresponding gene (for example,with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes are encompassed bythe present invention.

A genomic DNA or cDNA library prepared from such other strains may,therefore, be screened for DNA that hybridizes with the probes describedabove and encodes a polypeptide having aspartic endopeptidase activity.Genomic or other DNA from such other strains may be separated by agaroseor polyacrylamide gel electrophoresis, or other separation techniques.DNA from the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA that is homologous with SEQ ID NO: 1,or a subsequence thereof, the carrier material is preferably used in aSouthern blot.

For purposes of the present invention, hybridization indicates that thenucleotide sequence hybridizes to a labeled nucleic acid probecorresponding to the mature polypeptide coding sequence of SEQ ID NO: 1;the genomic DNA sequence comprising the mature polypeptide codingsequence of SEQ ID NO: 1; its full-length complementary strand; or asubsequence thereof; under very low to very high stringency conditions.Molecules to which the nucleic acid probe hybridizes under theseconditions can be detected using, for example, X-ray film.

In a preferred aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 1. In another preferred aspect, thenucleic acid probe is nucleotides 61 to 1401 of SEQ ID NO: 1. In anotherpreferred aspect, the nucleic acid probe is a polynucleotide sequencethat encodes the polypeptide of SEQ ID NO: 2, or a subsequence thereof.In another preferred aspect, the nucleic acid probe is SEQ ID NO: 1. Inanother preferred aspect, the nucleic acid probe is the polynucleotidesequence contained in plasmid pTter30A5 which is contained in E. coliNRRL B-50209, wherein the polynucleotide sequence thereof encodes apolypeptide having aspartic endopeptidase activity. In another preferredaspect, the nucleic acid probe is the mature polypeptide coding regioncontained in plasmid pTter30A5 which is contained in E. coli NRRLB-50209.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for very low andlow stringencies, 35% formamide for medium and medium-high stringencies,or 50% formamide for high and very high stringencies, following standardSouthern blotting procedures for 12 to 24 hours optimally.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at 45° C. (very low stringency), more preferably at50° C. (low stringency), more preferably at 55° C. (medium stringency),more preferably at 60° C. (medium-high stringency), even more preferablyat 65° C. (high stringency), and most preferably at 70° C. (very highstringency).

For short probes of about 15 nucleotides to about 70 nucleotides inlength, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at about 5° C. to about10° C. below the calculated T_(m) using the calculation according toBolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures for 12 to 24 hoursoptimally.

For short probes of about 15 nucleotides to about 70 nucleotides inlength, the carrier material is washed once in 6×SCC plus 0.1% SDS for15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10° C.below the calculated T_(m).

In a third aspect, the present invention relates to isolatedpolypeptides having aspartic endopeptidase activity encoded bypolynucleotides comprising or consisting of nucleotide sequences havinga degree of sequence identity to the mature polypeptide coding sequenceof SEQ ID NO: 1 of preferably at least 60%, more preferably at least65%, more preferably at least 70%, more preferably at least 75%, morepreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which encode a polypeptide having aspartic endopeptidase activity. Seepolynucleotide section herein.

In a fourth aspect, the present invention relates to artificial variantscomprising a substitution, deletion, and/or insertion of one or more (orseveral) amino acids of the mature polypeptide of SEQ ID NO: 2, or ahomologous sequence thereof. Preferably, amino acid changes are of aminor nature, that is conservative amino acid substitutions orinsertions that do not significantly affect the folding and/or activityof the protein; small deletions, typically of one to about 30 aminoacids; small amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue; a small linker peptide of up to about20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

In addition to the 20 standard amino acids, non-standard amino acids(such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid,isovaline, and alpha-methyl serine) may be substituted for amino acidresidues of a wild-type polypeptide. A limited number ofnon-conservative amino acids, amino acids that are not encoded by thegenetic code, and unnatural amino acids may be substituted for aminoacid residues. “Unnatural amino acids” have been modified after proteinsynthesis, and/or have a chemical structure in their side chain(s)different from that of the standard amino acids. Unnatural amino acidscan be chemically synthesized, and are preferably commerciallyavailable, and include pipecolic acid, thiazolidine carboxylic acid,dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in the parent polypeptide can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (i.e.,aspartic endopeptidase activity) to identify amino acid residues thatare critical to the activity of the molecule. See also, Hilton et al.,1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzyme orother biological interaction can also be determined by physical analysisof structure, as determined by such techniques as nuclear magneticresonance, crystallography, electron diffraction, or photoaffinitylabeling, in conjunction with mutation of putative contact site aminoacids. See, for example, de Vos et al., 1992, Science 255: 306-312;Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992,FEBS Lett. 309: 59-64. The identities of essential amino acids can alsobe inferred from analysis of identities with polypeptides that arerelated to a polypeptide according to the invention.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochem. 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide of interest, and can be applied to polypeptides of unknownstructure.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 2 is 10, preferably9, more preferably 8, more preferably 7, more preferably at most 6, morepreferably 5, more preferably 4, even more preferably 3, most preferably2, and even most preferably 1.

Sources of Polypeptides Having Aspartic Endopeptidase Activity

A polypeptide having aspartic endopeptidase activity of the presentinvention may be obtained from microorganisms of any genus. For purposesof the present invention, the term “obtained from” as used herein inconnection with a given source shall mean that the polypeptide encodedby a nucleotide sequence is produced by the source or by a strain inwhich the nucleotide sequence from the source has been inserted. In apreferred aspect, the polypeptide obtained from a given source issecreted extracellularly.

A polypeptide having aspartic endopeptidase activity of the presentinvention may be a bacterial polypeptide. For example, the polypeptidemay be a gram positive bacterial polypeptide such as a Bacillus,Streptococcus, Streptomyces, Staphylococcus, Enterococcus,Lactobacillus, Lactococcus, Clostridium, Geobacillus, or Oceanobacilluspolypeptide having aspartic endopeptidase activity, or a Gram negativebacterial polypeptide such as an E. coli, Pseudomonas, Salmonella,Campylobacter, Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter,Neisseria, or Ureaplasma polypeptide having aspartic endopeptidaseactivity.

In a preferred aspect, the polypeptide is a Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having aspartic endopeptidase activity.

In another preferred aspect, the polypeptide is a Streptococcusequisimilis, Streptococcus pyogenes, Streptococcus uberis, orStreptococcus equi subsp. Zooepidemicus polypeptide having asparticendopeptidase activity.

In another preferred aspect, the polypeptide is a Streptomycesachromogenes, Streptomyces avermitilis, Streptomyces coelicolor,Streptomyces griseus, or Streptomyces lividans polypeptide havingaspartic endopeptidase activity.

A polypeptide having aspartic endopeptidase activity of the presentinvention may also be a fungal polypeptide, and more preferably a yeastpolypeptide such as a Candida, Kluyveromyces, Pichia, Saccharomyces,Schizosaccharomyces, or Yarrowia polypeptide having asparticendopeptidase activity; or more preferably a filamentous fungalpolypeptide such as an Acremonium, Agaricus, Alternaria, Aspergillus,Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium,Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes,Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium,Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula,Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria polypeptide having asparticendopeptidase activity.

In a preferred aspect, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasfi, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis polypeptide having asparticendopeptidase activity.

In another preferred aspect, the polypeptide is an Acremoniumcellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillusfumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillusnidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporiumkeratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum,Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium zonatum, Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicolainsolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum,Penicillium purpurogenum, Phanerochaete chrysosporium, Thielaviaachromatica, Thielavia albomyces, Thielavia albopilosa, Thielaviaaustraleinsis, Thielavia fimeti, Thielavia microspora, Thielaviaovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa,Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride polypeptide having aspartic endopeptidaseactivity.

In another preferred aspect, the polypeptide is a Thielavia achromatica,Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis,Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielaviaperuviana, Thielavia spededonium, Thielavia setosa, Thielaviasubthermophila, or Thielavia terrestris polypeptide

In a more preferred aspect, the polypeptide is a Thielavia terrestrispolypeptide having catalase activity. In a most preferred aspect, thepolypeptide is a Thielavia terrestris NRRL 8126 polypeptide havingcatalase activity, e.g., the polypeptide comprising SEQ ID NO: 2.

It will be understood that for the aforementioned species the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms from natural habitats are well known in theart. The polynucleotide may then be obtained by similarly screening agenomic or cDNA library of such a microorganism. Once a polynucleotideencoding a polypeptide has been detected with the probe(s), thepolynucleotide can be isolated or cloned by utilizing techniques thatare well known to those of ordinary skill in the art (see, e.g.,Sambrook et al., 1989, supra).

Polypeptides of the present invention also include fused polypeptides orcleavable fusion polypeptides in which another polypeptide is fused atthe N-terminus or the C-terminus of the polypeptide or fragment thereof.A fused polypeptide is produced by fusing a nucleotide sequence (or aportion thereof) encoding another polypeptide to a nucleotide sequence(or a portion thereof) of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fused polypeptide is under control of thesame promoter(s) and terminator.

A fusion polypeptide can further comprise a cleavage site. Uponsecretion of the fusion protein, the site is cleaved releasing thepolypeptide having aspartic endopeptidase activity from the fusionprotein. Examples of cleavage sites include, but are not limited to, aKex2 site that encodes the dipeptide Lys-Arg (Martin et al., 2003, J.Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J.Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ.Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503;and Contreras et al., 1991, Biotechnology 9: 378-381), an Ile-(Glu orAsp)-Gly-Arg site, which is cleaved by a Factor Xa protease after thearginine residue (Eaton et al., 1986, Biochem. 25: 505-512); aAsp-Asp-Asp-Asp-Lys site, which is cleaved by an enterokinase after thelysine (Collins-Racie et al., 1995, Biotechnology 13: 982-987); aHis-Tyr-Glu site or His-Tyr-Asp site, which is cleaved by Genenase I(Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:240-248); a Leu-Val-Pro-Arg-Gly-Ser site, which is cleaved by thrombinafter the Arg (Stevens, 2003, Drug Discovery World 4: 35-48); aGlu-Asn-Leu-Tyr-Phe-Gln-Gly site, which is cleaved by TEV protease afterthe Gln (Stevens, 2003, supra); and a Leu-Glu-Val-Leu-Phe-Gln-Gly-Prosite, which is cleaved by a genetically engineered form of humanrhinovirus 3C protease after the Gln (Stevens, 2003, supra).

Polynucleotides

The present invention also relates to isolated polynucleotidescomprising or consisting of nucleotide sequences that encodepolypeptides having aspartic endopeptidase activity of the presentinvention.

In a preferred aspect, the nucleotide sequence comprises or consists ofSEQ ID NO: 1. In another more preferred aspect, the nucleotide sequencecomprises or consists of the sequence contained in plasmid pTter30A5which is contained in E. coli NRRL B-50209. In another preferred aspect,the nucleotide sequence comprises or consists of the mature polypeptidecoding sequence of SEQ ID NO: 1. In another preferred aspect, thenucleotide sequence comprises or consists of nucleotides 61 to 1401 ofSEQ ID NO: 1. In another more preferred aspect, the nucleotide sequencecomprises or consists of the mature polypeptide coding sequencecontained in plasmid pTter30A5 which is contained in E. coli NRRLB-50209. The present invention also encompasses nucleotide sequencesthat encode polypeptides comprising or consisting of the amino acidsequence of SEQ ID NO: 2 or the mature polypeptide thereof, which differfrom SEQ ID NO: 1 or the mature polypeptide coding sequence thereof byvirtue of the degeneracy of the genetic code. The present invention alsorelates to subsequences of SEQ ID NO: 1 that encode fragments of SEQ IDNO: 2 having aspartic endopeptidase activity.

The present invention also relates to mutant polynucleotides comprisingor consisting of at least one mutation in the mature polypeptide codingsequence of SEQ ID NO: 1, in which the mutant nucleotide sequenceencodes the mature polypeptide of SEQ ID NO: 2.

The techniques used to isolate or clone a polynucleotide encoding apolypeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thepolynucleotides of the present invention from such genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleotidesequence-based amplification (NASBA) may be used. The polynucleotidesmay be cloned from a strain of Thielavia, or another or related organismand thus, for example, may be an allelic or species variant of thepolypeptide encoding region of the nucleotide sequence.

The present invention also relates to isolated polynucleotidescomprising or consisting of nucleotide sequences having a degree ofsequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 of preferably at least 60%, more preferably at least 65%, morepreferably at least 70%, more preferably at least 75%, more preferablyat least 80%, more preferably at least 85%, even more preferably atleast 90%, most preferably at least 95%, and even most preferably atleast 96%, at least 97%, at least 98%, or at least 99%, which encode apolypeptide having aspartic endopeptidase activity.

Modification of a nucleotide sequence encoding a polypeptide of thepresent invention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source, e.g., artificialvariants that differ in specific activity, thermostability, pH optimum,or the like. The variant sequence may be constructed on the basis of thenucleotide sequence presented as the mature polypeptide coding sequenceof SEQ ID NO: 1, e.g., a subsequence thereof, and/or by introduction ofnucleotide substitutions that do not give rise to another amino acidsequence of the polypeptide encoded by the nucleotide sequence, butwhich correspond to the codon usage of the host organism intended forproduction of the enzyme, or by introduction of nucleotide substitutionsthat may give rise to a different amino acid sequence. For a generaldescription of nucleotide substitution, see, e.g., Ford et al., 1991,Protein Expression and Purification 2: 95-107.

It will be apparent to those skilled in the art that such substitutionscan be made outside the regions critical to the function of the moleculeand still result in an active polypeptide. Amino acid residues essentialto the activity of the polypeptide encoded by an isolated polynucleotideof the invention, and therefore preferably not subject to substitution,may be identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (see, e.g.,Cunningham and Wells, 1989, supra). In the latter technique, mutationsare introduced at every positively charged residue in the molecule, andthe resultant mutant molecules are tested for aspartic endopeptidaseactivity to identify amino acid residues that are critical to theactivity of the molecule. Sites of substrate-enzyme interaction can alsobe determined by analysis of the three-dimensional structure asdetermined by such techniques as nuclear magnetic resonance analysis,crystallography or photoaffinity labeling (see, e.g., de Vos et al.,1992, supra; Smith et al., 1992, supra; Wlodaver et al., 1992, supra).

The present invention also relates to isolated polynucleotides encodingpolypeptides of the present invention, which hybridize under preferablyvery low stringency conditions, more preferably low stringencyconditions, more preferably medium stringency conditions, morepreferably medium-high stringency conditions, even more preferably highstringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the genomic DNA sequence comprising the mature polypeptidecoding sequence of SEQ ID NO: 1, or (iii) a full-length complementarystrand of (i) or (ii); or allelic variants and subsequences thereof(Sambrook et al., 1989, supra), as defined herein.

The present invention also relates to isolated polynucleotides obtainedby (a) hybridizing a population of DNA under preferably very lowstringency conditions, more preferably low stringency conditions, morepreferably medium stringency conditions, more preferably medium-highstringency conditions, even more preferably high stringency conditions,and most preferably very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 1, (ii) the genomic DNAsequence comprising the mature polypeptide coding sequence of SEQ ID NO:1, or (iii) a full-length complementary strand of (i) or (ii); and (b)isolating the hybridizing polynucleotide, which encodes a polypeptidehaving aspartic endopeptidase activity.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisingan isolated polynucleotide of the present invention operably linked toone or more (several) control sequences that direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences.

An isolated polynucleotide encoding a polypeptide of the presentinvention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide'ssequence prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotide sequences utilizing recombinant DNA methods arewell known in the art.

The control sequence may be an appropriate promoter sequence, anucleotide sequence that is recognized by a host cell for expression ofa polynucleotide encoding a polypeptide of the present invention. Thepromoter sequence contains transcriptional control sequences thatmediate the expression of the polypeptide. The promoter may be anynucleotide sequence that shows transcriptional activity in the host cellof choice including mutant, truncated, and hybrid promoters, and may beobtained from genes encoding extracellular or intracellular polypeptideseither homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xyIA and xyIBgenes, and prokaryotic beta-lactamase gene (VIIIa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporumtrypsin-like protease (WO 96/00787), Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as a NA2-tpipromoter (a modified promoter including a gene encoding a neutralalpha-amylase in Aspergilli in which the untranslated leader has beenreplaced by an untranslated leader from a gene encoding triose phosphateisomerase in Aspergilli; non-limiting examples include modifiedpromoters including the gene encoding neutral alpha-amylase inAspergillus niger in which the untranslated leader has been replaced byan untranslated leader from the gene encoding triose phosphate isomerasein Aspergillus nidulans or Aspergillus oryzae); and mutant, truncated,and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleotide sequence encoding the polypeptide. Anyterminator that is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA that is important for translation by thehost cell. The leader sequence is operably linked to the 5′ terminus ofthe nucleotide sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleotide sequence and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding sequence thatencodes a signal peptide linked to the amino terminus of a polypeptideand directs the encoded polypeptide into the cell's secretory pathway.The 5′ end of the coding sequence of the nucleotide sequence mayinherently contain a signal peptide coding sequence naturally linked intranslation reading frame with the segment of the coding sequence thatencodes the secreted polypeptide. Alternatively, the 5′ end of thecoding sequence may contain a signal peptide coding sequence that isforeign to the coding sequence. The foreign signal peptide codingsequence may be required where the coding sequence does not naturallycontain a signal peptide coding sequence. Alternatively, the foreignsignal peptide coding sequence may simply replace the natural signalpeptide coding sequence in order to enhance secretion of thepolypeptide. However, any signal peptide coding sequence that directsthe expressed polypeptide into the secretory pathway of a host cell ofchoice, i.e., secreted into a culture medium, may be used in the presentinvention.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus stearothermophilusalpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, Humicola insolens endoglucanase V, andHumicola lanuginosa lipase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

In a preferred aspect, the signal peptide comprises or consists of aminoacids 1 to 20 of SEQ ID NO: 2. In another preferred aspect, the signalpeptide coding sequence comprises or consists of nucleotides 1 to 60 ofSEQ ID NO: 1.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the amino terminus of a polypeptide.The resultant polypeptide is known as a proenzyme or propolypeptide (ora zymogen in some cases). A propeptide is generally inactive and can beconverted to a mature active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei asparticproteinase, and Myceliophthora thermophila laccase (WO 95/33836).

Where both signal peptide and propeptide sequences are present at theamino terminus of a polypeptide, the propeptide sequence is positionednext to the amino terminus of a polypeptide and the signal peptidesequence is positioned next to the amino terminus of the propeptidesequence.

It may also be desirable to add regulatory sequences that allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those that causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those that allow for geneamplification. In eukaryotic systems, these regulatory sequences includethe dihydrofolate reductase gene that is amplified in the presence ofmethotrexate, and the metallothionein genes that are amplified withheavy metals. In these cases, the nucleotide sequence encoding thepolypeptide would be operably linked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleicacids and control sequences described herein may be joined together toproduce a recombinant expression vector that may include one or more(several) convenient restriction sites to allow for insertion orsubstitution of the nucleotide sequence encoding the polypeptide at suchsites. Alternatively, a polynucleotide sequence of the present inventionmay be expressed by inserting the nucleotide sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the nucleotide sequence. The choice ofthe vector will typically depend on the compatibility of the vector withthe host cell into which the vector is to be introduced. The vectors maybe linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vectors of the present invention preferably contain one or more(several) selectable markers that permit easy selection of transformed,transfected, transduced, or the like cells. A selectable marker is agene the product of which provides for biocide or viral resistance,resistance to heavy metals, prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers that confer antibioticresistance such as ampicillin, kanamycin, chloramphenicol, ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits integration of the vector into the host cell's genome orautonomous replication of the vector in the cell independent of thegenome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornonhomologous recombination. Alternatively, the vector may containadditional nucleotide sequences for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 10,000 base pairs,preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000base pairs, which have a high degree of sequence identity to thecorresponding target sequence to enhance the probability of homologousrecombination. The integrational elements may be any sequence that ishomologous with the target sequence in the genome of the host cell.Furthermore, the integrational elements may be non-encoding or encodingnucleotide sequences. On the other hand, the vector may be integratedinto the genome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” is definedherein as a nucleotide sequence that enables a plasmid or vector toreplicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMR1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation ofthe AMA1 gene and construction of plasmids or vectors comprising thegene can be accomplished according to the methods disclosed in WO00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of the gene product. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisingan isolated polynucleotide of the present invention operably linked toone or more (several) control sequences that direct the production of apolypeptide having aspartic endopeptidase activity. A construct orvector comprising a polynucleotide of the present invention isintroduced into a host cell so that the construct or vector ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thepolypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram positive bacterium or a Gramnegative bacterium. Gram positive bacteria include, but not limited to,Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus,Lactobacillus, Lactococcus, Clostridium, Geobacillus, andOceanobacillus. Gram negative bacteria include, but not limited to, E.coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter,Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, and Ureaplasma.

The bacterial host cell may be any Bacillus cell. Bacillus cells usefulin the practice of the present invention include, but are not limitedto, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillusfirmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis,Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus,Bacillus subtilis, and Bacillus thuringiensis cells.

In a preferred aspect, the bacterial host cell is a Bacillusamyloliquefaciens cell. In another preferred aspect, the bacterial hostcell is a Bacillus clausii cell. In another preferred aspect, thebacterial host cell is a Bacillus lentus cell. In another preferredaspect, the bacterial host cell is a Bacillus licheniformis cell. Inanother preferred aspect, the bacterial host cell is a Bacillusstearothermophilus cell. In another preferred aspect, the bacterial hostcell is a Bacillus subtilis cell.

The bacterial host cell may also be any Streptococcus cell.Streptococcus cells useful in the practice of the present inventioninclude, but are not limited to, Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equisubsp. Zooepidemicus cells.

In a preferred aspect, the bacterial host cell is a Streptococcusequisimilis cell. In another preferred aspect, the bacterial host cellis a Streptococcus pyogenes cell. In another preferred aspect, thebacterial host cell is a Streptococcus uberis cell. In another preferredaspect, the bacterial host cell is a Streptococcus equi subsp.Zooepidemicus cell.

The bacterial host cell may also be any Streptomyces cell. Streptomycescells useful in the practice of the present invention include, but arenot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

In a preferred aspect, the bacterial host cell is a Streptomycesachromogenes cell. In another preferred aspect, the bacterial host cellis a Streptomyces avermitilis cell. In another preferred aspect, thebacterial host cell is a Streptomyces coelicolor cell. In anotherpreferred aspect, the bacterial host cell is a Streptomyces griseuscell. In another preferred aspect, the bacterial host cell is aStreptomyces lividans cell.

The introduction of DNA into a Bacillus cell may, for instance, beeffected by protoplast transformation (see, e.g., Chang and Cohen, 1979,Molecular General Genetics 168: 111-115), by using competent cells (see,e.g., Young and Spizizen, 1961, Journal of Bacteriology 81: 823-829, orDubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56:209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988,Biotechniques 6: 742-751), or by conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5271-5278). The introductionof DNA into an E. coli cell may, for instance, be effected by protoplasttransformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-580) orelectroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16:6127-6145). The introduction of DNA into a Streptomyces cell may, forinstance, be effected by protoplast transformation and electroporation(see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405), byconjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or by transduction (see, e.g., Burke et al., 2001, Proc.Natl. Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may, for instance, be effected by electroporation (see,e.g., Choi et al., 2006, J. Microbiol. Methods 64: 391-397) or byconjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ.Microbiol. 71: 51-57). The introduction of DNA into a Streptococcus cellmay, for instance, be effected by natural competence (see, e.g., Perryand Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), by protoplasttransformation (see, e.g., Catt and Jollick, 1991, Microbios. 68:189-207, by electroporation (see, e.g., Buckley et al., 1999, Appl.Environ. Microbiol. 65: 3800-3804) or by conjugation (see, e.g.,Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any method knownin the art for introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

In a preferred aspect, the host cell is a fungal cell. “Fungi” as usedherein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota,and Zygomycota (as defined by Hawksworth et al., In, Ainsworth andBisby's Dictionary of The Fungi, 8th edition, 1995, CAB International,University Press, Cambridge, UK) as well as the Oomycota (as cited inHawksworth et al., 1995, supra, page 171) and all mitosporic fungi(Hawksworth et al., 1995, supra).

In a more preferred aspect, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

In an even more preferred aspect, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.

In a most preferred aspect, the yeast host cell is a Saccharomycescarlsbergensis cell. In another most preferred aspect, the yeast hostcell is a Saccharomyces cerevisiae cell. In another most preferredaspect, the yeast host cell is a Saccharomyces diastaticus cell. Inanother most preferred aspect, the yeast host cell is a Saccharomycesdouglasii cell. In another most preferred aspect, the yeast host cell isa Saccharomyces kluyveri cell. In another most preferred aspect, theyeast host cell is a Saccharomyces norbensis cell. In another mostpreferred aspect, the yeast host cell is a Saccharomyces oviformis cell.In another most preferred aspect, the yeast host cell is a Kluyveromyceslactis cell. In another most preferred aspect, the yeast host cell is aYarrowia lipolytica cell.

In another more preferred aspect, the fungal host cell is a filamentousfungal cell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are generally characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative.

In an even more preferred aspect, the filamentous fungal host cell is anAcremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium,Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix,Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia,Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus,Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

In a most preferred aspect, the filamentous fungal host cell is anAspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger orAspergillus oryzae cell. In another most preferred aspect, thefilamentous fungal host cell is a Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusariumvenenatum cell. In another most preferred aspect, the filamentous fungalhost cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsisaneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens,Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,Ceriporiopsis subvermispora, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium tropicum, Chrysosporium merdarium,Chrysosporium inops, Chrysosporium pannicola, Chrysosporiumqueenslandicum, Chrysosporium zonatum, Coprinus cinereus, Coriolushirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

In another most preferred aspect, the filamentous fungal host cell is anAspergillus niger cell. In another most preferred aspect, thefilamentous fungal host cell is an Aspergillus oryzae cell. In anothermost preferred aspect, the filamentous fungal host cell is aChrysosporium lucknowense cell. In another most preferred aspect, thefilamentous fungal host cell is a Fusarium venenatum cell. In anothermost preferred aspect, the filamentous fungal host cell is aMyceliophthora thermophila cell. In another most preferred aspect, thefilamentous fungal host cell is a Trichoderma reesei cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238 023 and Yelton et al., 1984, Proceedings of the NationalAcademy of Sciences USA 81: 1470-1474. Suitable methods for transformingFusarium species are described by Malardier et al., 1989, Gene 78:147-156, and WO 96/00787. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,editors, Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Itoet al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a cell, which inits wild-type form produces the polypeptide, under conditions conducivefor production of the polypeptide; and (b) recovering the polypeptide.In a preferred aspect, the cell is of the genus Thielavia. In a morepreferred aspect, the cell is Thielavia terrestris. In a most preferredaspect, the cell is Thielavia terrestris NRRL 8126.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a recombinant hostcell, as described herein, under conditions conducive for production ofthe polypeptide; and (b) recovering the polypeptide.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a recombinant hostcell under conditions conducive for production of the polypeptide,wherein the host cell comprises a mutant nucleotide sequence having atleast one mutation in the mature polypeptide coding sequence of SEQ IDNO: 1, wherein the mutant nucleotide sequence encodes a polypeptide thatcomprises or consists of the mature polypeptide of SEQ ID NO: 2; and (b)recovering the polypeptide.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods well known in the art. For example, the cellmay be cultivated by shake flask cultivation, and small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted into the medium, it can be recovered fromcell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide as described herein.

The resulting polypeptide may be recovered using methods known in theart. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989) to obtain substantially pure polypeptides.

Plants

The present invention also relates to plants, e.g., a transgenic plant,plant part, or plant cell, comprising an isolated polynucleotideencoding a polypeptide having aspartic endopeptidase activity of thepresent invention so as to express and produce the polypeptide inrecoverable quantities. The polypeptide may be recovered from the plantor plant part. Alternatively, the plant or plant part containing therecombinant polypeptide may be used as such for improving the quality ofa food or feed, e.g., improving nutritional value, palatability, andrheological properties, or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilisation of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seeds coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a polypeptide of thepresent invention may be constructed in accordance with methods known inthe art. In short, the plant or plant cell is constructed byincorporating one or more (several) expression constructs encoding apolypeptide of the present invention into the plant host genome orchloroplast genome and propagating the resulting modified plant or plantcell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a polypeptide of the presentinvention operably linked with appropriate regulatory sequences requiredfor expression of the nucleotide sequence in the plant or plant part ofchoice. Furthermore, the expression construct may comprise a selectablemarker useful for identifying host cells into which the expressionconstruct has been integrated and DNA sequences necessary forintroduction of the construct into the plant in question (the latterdepends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide of the present invention may be constitutive or inducible,or may be developmental, stage or tissue specific, and the gene productmay be targeted to a specific tissue or plant part such as seeds orleaves. Regulatory sequences are, for example, described by Tague etal., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, andthe rice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhanget al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoterfrom the legumin B4 and the unknown seed protein gene from Vicia faba(Conrad et al., 1998, Journal of Plant Physiology 152: 708-711), apromoter from a seed oil body protein (Chen et al., 1998, Plant and CellPhysiology 39: 935-941), the storage protein napA promoter from Brassicanapus, or any other seed specific promoter known in the art, e.g., asdescribed in WO 91/14772. Furthermore, the promoter may be a leafspecific promoter such as the rbcs promoter from rice or tomato (Kyozukaet al., 1993, Plant Physiology 102: 991-1000, the chlorella virusadenine methyltransferase gene promoter (Mitra and Higgins, 1994, PlantMolecular Biology 26: 85-93), or the aldP gene promoter from rice(Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or awound inducible promoter such as the potato pin2 promoter (Xu et al.,1993, Plant Molecular Biology 22: 573-588). Likewise, the promoter mayinducible by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide of the present invention in the plant. Forinstance, the promoter enhancer element may be an intron that is placedbetween the promoter and the nucleotide sequence encoding a polypeptideof the present invention. For instance, Xu et al., 1993, supra, disclosethe use of the first intron of the rice actin 1 gene to enhanceexpression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38) andcan also be used for transforming monocots, although othertransformation methods are often used for these plants. Presently, themethod of choice for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant Journal 2: 275-281; Shimamoto, 1994, Current OpinionBiotechnology 5: 158-162; Vasil et al., 1992, Bio/Technology 10:667-674). An alternative method for transformation of monocots is basedon protoplast transformation as described by Omirulleh et al., 1993,Plant Molecular Biology 21: 415-428. Additional transformation methodsfor use in accordance with the present disclosure include thosedescribed in U.S. Pat. Nos. 6,395,966 and 7,151,204 (both of which areherein incorporated by reference in their entirety).

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well-known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

The present invention also relates to methods of producing a polypeptideof the present invention comprising: (a) cultivating a transgenic plantor a plant cell comprising a polynucleotide encoding the polypeptidehaving aspartic endopeptidase activity of the present invention underconditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

In embodiments, in addition to direct transformation of a particularplant genotype with a construct prepared according to the presentinvention, transgenic plants may be made by crossing a plant having aconstruct of the present invention to a second plant lacking theconstruct. For example, a construct encoding a polypeptide havingaspartic endopeptidase activity or a portion thereof can be introducedinto a particular plant variety by crossing, without the need for everdirectly transforming a plant of that given variety. Therefore, thepresent invention not only encompasses a plant directly regenerated fromcells which have been transformed in accordance with the presentinvention, but also the progeny of such plants. As used herein, progenymay refer to the offspring of any generation of a parent plant preparedin accordance with the present invention. Such progeny may include a DNAconstruct prepared in accordance with the present invention, or aportion of a DNA construct prepared in accordance with the presentinvention. In embodiments, crossing results in a transgene of thepresent invention being introduced into a plant line by crosspollinating a starting line with a donor plant line that includes atransgene of the present invention. Non-limiting examples of such stepsare further articulated in U.S. Pat. No. 7,151,204.

It is envisioned that plants including a polypeptide having asparticendopeptidase activity of the present invention include plants generatedthrough a process of backcross conversion. For examples, plants of thepresent invention include plants referred to as a backcross convertedgenotype, line, inbred, or hybrid.

In embodiments, genetic markers may be used to assist in theintrogression of one or more transgenes of the invention from onegenetic background into another. Marker assisted selection offersadvantages relative to conventional breeding in that it can be used toavoid errors caused by phenotypic variations. Further, genetic markersmay provide data regarding the relative degree of elite germplasm in theindividual progeny of a particular cross. For example, when a plant witha desired trait which otherwise has a non-agronomically desirablegenetic background is crossed to an elite parent, genetic markers may beused to select progeny which not only possess the trait of interest, butalso have a relatively large proportion of the desired germplasm. Inthis way, the number of generations required to introgress one or moretraits into a particular genetic background is minimized.

Removal or Reduction of Aspartic Endopeptidase Activity

The present invention also relates to methods of producing a mutant of aparent cell, which comprises disrupting or deleting a polynucleotide, ora portion thereof, encoding a polypeptide of the present invention,which results in the mutant cell producing less of the polypeptide thanthe parent cell when cultivated under the same conditions.

The mutant cell may be constructed by reducing or eliminating expressionof a nucleotide sequence encoding a polypeptide of the present inventionusing methods well known in the art, for example, insertions,disruptions, replacements, or deletions. In a preferred aspect, thenucleotide sequence is inactivated. The nucleotide sequence to bemodified or inactivated may be, for example, the coding region or a partthereof essential for activity, or a regulatory element required for theexpression of the coding region. An example of such a regulatory orcontrol sequence may be a promoter sequence or a functional partthereof, i.e., a part that is sufficient for affecting expression of thenucleotide sequence. Other control sequences for possible modificationinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, signal peptide sequence, transcription terminator,and transcriptional activator.

Modification or inactivation of the nucleotide sequence may be performedby subjecting the parent cell to mutagenesis and selecting for mutantcells in which expression of the nucleotide sequence has been reduced oreliminated. The mutagenesis, which may be specific or random, may beperformed, for example, by use of a suitable physical or chemicalmutagenizing agent, by use of a suitable oligonucleotide, or bysubjecting the DNA sequence to PCR generated mutagenesis. Furthermore,the mutagenesis may be performed by use of any combination of thesemutagenizing agents.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed byincubating the parent cell to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions, and screeningand/or selecting for mutant cells exhibiting reduced or no expression ofthe gene.

Modification or inactivation of the nucleotide sequence may beaccomplished by introduction, substitution, or removal of one or more(several) nucleotides in the gene or a regulatory element required forthe transcription or translation thereof. For example, nucleotides maybe inserted or removed so as to result in the introduction of a stopcodon, the removal of the start codon, or a change in the open readingframe. Such modification or inactivation may be accomplished bysite-directed mutagenesis or PCR generated mutagenesis in accordancewith methods known in the art. Although, in principle, the modificationmay be performed in vivo, i.e., directly on the cell expressing thenucleotide sequence to be modified, it is preferred that themodification be performed in vitro as exemplified below.

An example of a convenient way to eliminate or reduce expression of anucleotide sequence by a cell is based on techniques of genereplacement, gene deletion, or gene disruption. For example, in the genedisruption method, a nucleic acid sequence corresponding to theendogenous nucleotide sequence is mutagenized in vitro to produce adefective nucleic acid sequence that is then transformed into the parentcell to produce a defective gene. By homologous recombination, thedefective nucleic acid sequence replaces the endogenous nucleotidesequence. It may be desirable that the defective nucleotide sequencealso encodes a marker that may be used for selection of transformants inwhich the nucleotide sequence has been modified or destroyed. In aparticularly preferred aspect, the nucleotide sequence is disrupted witha selectable marker such as those described herein.

Alternatively, modification or inactivation of the nucleotide sequencemay be performed by established anti-sense or RNAi techniques using asequence complementary to the nucleotide sequence. More specifically,expression of the nucleotide sequence by a cell may be reduced oreliminated by introducing a sequence complementary to the nucleotidesequence of the gene that may be transcribed in the cell and is capableof hybridizing to the mRNA produced in the cell. Under conditionsallowing the complementary anti-sense nucleotide sequence to hybridizeto the mRNA, the amount of protein translated is thus reduced oreliminated.

The present invention further relates to a mutant cell of a parent cellthat comprises a disruption or deletion of a nucleotide sequenceencoding the polypeptide or a control sequence thereof, which results inthe mutant cell producing less of the polypeptide or no polypeptidecompared to the parent cell.

The polypeptide-deficient mutant cells so created are particularlyuseful as host cells for the expression of native and/or heterologouspolypeptides. Therefore, the present invention further relates tomethods of producing a native or heterologous polypeptide, comprising:(a) cultivating the mutant cell under conditions conducive forproduction of the polypeptide; and (b) recovering the polypeptide. Theterm “heterologous polypeptides” is defined herein as polypeptides thatare not native to the host cell, a native protein in which modificationshave been made to alter the native sequence, or a native protein whoseexpression is quantitatively altered as a result of a manipulation ofthe host cell by recombinant DNA techniques.

In a further aspect, the present invention relates to a method ofproducing a protein product essentially free of aspartic endopeptidaseactivity by fermentation of a cell that produces both a polypeptide ofthe present invention as well as the protein product of interest byadding an effective amount of an agent capable of inhibiting asparticendopeptidase activity to the fermentation broth before, during, orafter the fermentation has been completed, recovering the product ofinterest from the fermentation broth, and optionally subjecting therecovered product to further purification.

In a further aspect, the present invention relates to a method ofproducing a protein product essentially free of aspartic endopeptidaseactivity by cultivating the cell under conditions permitting theexpression of the product, subjecting the resultant culture broth to acombined pH and temperature treatment so as to reduce the asparticendopeptidase activity substantially, and recovering the product fromthe culture broth. Alternatively, the combined pH and temperaturetreatment may be performed on an enzyme preparation recovered from theculture broth. The combined pH and temperature treatment may optionallybe used in combination with a treatment with an aspartic endopeptidaseinhibitor.

In accordance with this aspect of the invention, it is possible toremove at least 60%, preferably at least 75%, more preferably at least85%, still more preferably at least 95%, and most preferably at least99% of the aspartic endopeptidase activity. Complete removal of asparticendopeptidase activity may be obtained by use of this method.

The combined pH and temperature treatment is preferably carried out at apH in the range of 2-4 or 9-11 and a temperature in the range of atleast 60-70° C. for a sufficient period of time to attain the desiredeffect, where typically, 30 to 60 minutes is sufficient.

The methods used for cultivation and purification of the product ofinterest may be performed by methods known in the art.

The methods of the present invention for producing an essentiallyaspartic endopeptidase-free product is of particular interest in theproduction of eukaryotic polypeptides, in particular fungal proteinssuch as enzymes. The enzyme may be selected from, e.g., an amylolyticenzyme, lipolytic enzyme, proteolytic enzyme, cellulolytic enzyme,oxidoreductase, or plant cell-wall degrading enzyme. Examples of suchenzymes include an aminopeptidase, amylase, amyloglucosidase,carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase,chitinase, cutinase, cyclodextrin glycosyltransferase,deoxyribonuclease, endoglucanase, esterase, galactosidase,beta-galactosidase, glucoamylase, glucose oxidase, glucosidase,haloperoxidase, hemicellulase, invertase, isomerase, laccase, ligase,lipase, lyase, mannosidase, oxidase, pectinolytic enzyme, peroxidase,phytase, phenoloxidase, polyphenoloxidase, proteolytic enzyme,ribonuclease, transferase, transglutaminase, or xylanase. The asparticendopeptidase-deficient cells may also be used to express heterologousproteins of pharmaceutical interest such as hormones, growth factors,receptors, and the like.

It will be understood that the term “eukaryotic polypeptides” includesnot only native polypeptides, but also those polypeptides, e.g.,enzymes, which have been modified by amino acid substitutions, deletionsor additions, or other such modifications to enhance activity,thermostability, pH tolerance and the like.

In a further aspect, the present invention relates to a protein productessentially free from aspartic endopeptidase activity that is producedby a method of the present invention.

Methods of Inhibiting Expression of a Polypeptide Having AsparticEndopeptidase Activity

The present invention also relates to methods of inhibiting theexpression of a polypeptide having aspartic endopeptidase activity in acell, comprising administering to the cell or expressing in the cell adouble-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises asubsequence of a polynucleotide of the present invention. In a preferredaspect, the dsRNA is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 ormore duplex nucleotides in length.

The dsRNA is preferably a small interfering RNA (siRNA) or a micro RNA(miRNA). In a preferred aspect, the dsRNA is small interfering RNA(siRNAs) for inhibiting transcription. In another preferred aspect, thedsRNA is micro RNA (miRNAs) for inhibiting translation.

The present invention also relates to such double-stranded RNA (dsRNA)molecules, comprising a portion of the mature polypeptide codingsequence of SEQ ID NO: 1 for inhibiting expression of a polypeptidehaving aspartic endopeptidase activity in a cell. While the presentinvention is not limited by any particular mechanism of action, thedsRNA can enter a cell and cause the degradation of a single-strandedRNA (ssRNA) of similar or identical sequences, including endogenousmRNAs. When a cell is exposed to dsRNA, mRNA from the homologous gene isselectively degraded by a process called RNA interference (RNAi).

The dsRNAs of the present invention can be used in gene-silencing. Inone aspect, the invention provides methods to selectively degrade RNAusing the dsRNAis of the present invention. The process may be practicedin vitro, ex vivo or in vivo. In one aspect, the dsRNA molecules can beused to generate a loss-of-function mutation in a cell, an organ or ananimal. Methods for making and using dsRNA molecules to selectivelydegrade RNA are well known in the art; see, for example, U.S. Pat. No.6,506,559; U.S. Pat. No. 6,511,824; U.S. Pat. No. 6,515,109; and U.S.Pat. No. 6,489,127.

Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. Preferably, the compositions areenriched in such a polypeptide. The term “enriched” indicates that theaspartic endopeptidase activity of the composition has been increased,e.g., with an enrichment factor of at least 1.1.

The composition may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the composition may comprise multiple enzymaticactivities, such as an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase,beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase,haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase,pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase,polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase,or xylanase. The additional enzyme(s) may be produced, for example, by amicroorganism belonging to the genus Aspergillus, preferably Aspergillusaculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger, or Aspergillus oryzae; Fusarium, preferably Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides, orFusarium venenatum; Humicola, preferably Humicola insolens or Humicolalanuginosa; or Trichoderma, preferably Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art.

Examples are given below of preferred uses of the polypeptidecompositions of the invention. The dosage of the polypeptide compositionof the invention and other conditions under which the composition isused may be determined on the basis of methods known in the art.

Uses

The present invention is also directed to methods for using thepolypeptides having aspartic endopeptidase activity, or compositionsthereof.

A polypeptide of the present invention may also be used in the food andfeed industry to improve the digestibility of proteins. For instance,the polypeptide may be added to animal feed or may be used to processanimal feed to improve the digestibility of components of the feed.

A polypeptide of the present invention may be useful to make proteinhydrolysates from, e.g., vegetable proteins like soy, pea, milk-likecasein, meat proteins, or fish proteins. The polypeptide may be used toimprove the solubility, consistency, taste, or fermentability, to reduceantigenicity or for other purposes to make food or feed products. Thepolypeptide may be used alone or together with other proteases ortogether with other enzymes like exopeptidases. The use of thepolypeptide of the present invention together with exopeptidase-richenzyme preparations will improve the taste of the protein hydrolysates.See, for example, WO 1998/014599, WO 1998/051803, WO 1998/051163, and WO1998/051804.

A polypeptide of the present invention may also be used to modifyproteins to reduce viscosity.

A polypeptide of the present invention may also be used in low pHbaking.

A polypeptide of the present invention may also be used as milkcoagulants (see WO 2003/206484)

For the above uses, the dosage of the polypeptide of the presentinvention and other conditions under which the preparation is used maybe determined on the basis of methods known in the art.

Signal Peptide

The present invention also relates to an isolated polynucleotideencoding a signal peptide comprising or consisting of amino acids 1 to20 of SEQ ID NO: 2.

The present invention also relates to nucleic acid constructs comprisinga gene encoding a protein, wherein the gene is operably linked to theisolated polynucleotide encoding the signal peptide, wherein the gene isforeign to the polynucleotide encoding the signal peptide.

In a preferred aspect, the isolated polynucleotide encoding a signalpeptide comprises or consists of nucleotides 1 to 60 of SEQ ID NO: 1.

The present invention also relates to recombinant expression vectors andrecombinant host cells comprising such nucleic acid constructs.

The present invention also relates to methods of producing a protein,comprising: (a) cultivating a recombinant host cell comprising a geneencoding a protein operably linked to such a polynucleotide encoding asignal peptide, wherein the gene is foreign to the polynucleotideencoding the signal peptide, under conditions conducive for productionof the protein; and (b) recovering the protein.

The protein may be native or heterologous to a host cell. The term“protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andproteins. The term “protein” also encompasses two or more polypeptidescombined to form the encoded product. The proteins also include hybridpolypeptides that comprise a combination of partial or completepolypeptide sequences obtained from at least two different proteinswherein one or more (several) may be heterologous or native to the hostcell. Proteins further include naturally occurring allelic andengineered variations of the above mentioned proteins and hybridproteins.

Preferably, the protein is a hormone or variant thereof, enzyme,receptor or portion thereof, antibody or portion thereof, or reporter.In a more preferred aspect, the protein is an oxidoreductase,transferase, hydrolase, lyase, isomerase, or ligase. In an even morepreferred aspect, the protein is an aminopeptidase, amylase,carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, invertase, laccase, another lipase,mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase or xylanase.

The gene may be obtained from any prokaryotic, eukaryotic, or othersource.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Materials

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Strains

Thielavia terrestris NRRL 8126 was used as the source of a gene encodinga polypeptide having aspartic endopeptidase activity.

Media

PDA plates were composed of 39 grams of potato dextrose agar anddeionized water to 1 liter.

DAP2C-1 medium was composed of 1 g of KH₂PO₄, 11 g of MgSO₄.7H₂O, 2 g ofcitric acid, 30 g of maltodextrin, 6 g of K₃PO₄.3H₂O, 0.5 g of yeastextract, 0.5 ml of trace metals solution, 1 ml of Pluronic PE 6100, 1.66g of CaCO₃, and deionized water to 1 liter. After autoclaving, 23 ml of50% w/v (NH₄)₂HPO₄ and 33 ml of 20% lactic acid, both filter sterilized,were added.

Trace metals solution was composed of 6.8 g of ZnCl₂, 2.5 g ofCuSO₄.5H₂O, 0.24 g of NiCl₂.6H₂O, 13.9 g of FeSO₄.7H₂O, 8.45 g ofMnSO₄.H₂O, 3 g citric acid, and deionized water to 1 liter.

LB plates were composed of 10 g of tryptone, 5 g of yeast extract, 5 gof sodium chloride, 15 g of Bacto agar, and deionized water to 1 liter.

LB medium was composed of 10 g of tryptone, 5 g of yeast extract, 5 g ofsodium chloride, and deionized water to 1 liter.

SOC medium was composed of 2% tryptone, 0.5% yeast extract, 10 mM NaCl,2.5 mM KCl, 10 mM MgCl₂, and 10 mM MgSO₄, sterilized by autoclaving andthen filter-sterilized glucose was added to 20 mM.

Freezing medium was composed of 60% SOC medium and 40% glycerol.

Example 1 Expressed Sequence Tags (EST) cDNA Library Construction

Thielavia terrestris NRRL 8126 was cultivated in 50 ml of NNCYPmodmedium supplemented with 1% glucose in a 250 ml flask at 45° C. for 24hours with shaking at 200 rpm. A two ml aliquot from the 24-hour liquidculture was used to seed a 500 ml flask containing 100 ml of NNCYPmodmedium supplemented with 2% SIGMACELL® 20 (cellulose; Sigma ChemicalCo., Inc., St. Louis, Mo., USA). The culture was incubated at 45° C. for3 days with shaking at 200 rpm. The mycelia were harvested by filtrationthrough a funnel with a glass fiber prefilter (Nalgene, Rochester, N.Y.,USA), washed twice with 10 mM Tris-HCl-1 mM EDTA pH 8 (TE), and quickfrozen in liquid nitrogen.

Total RNA was isolated using the following method. Frozen mycelia ofThielavia terrestris NRRL 8126 were ground in an electric coffeegrinder. The ground material was mixed 1:1 v/v with 20 ml of FENAZOL™(Ambion, Inc., Austin, Tex., USA) in a 50 ml tube. Once the mycelia weresuspended, they were extracted with chloroform and three times with amixture of phenol-chloroform-isoamyl alcohol 25:24:1 v/v/v. From theresulting aqueous phase, the RNA was precipitated by adding 1/10 volumeof 3 M sodium acetate pH 5.2 and 1.25 volumes of isopropanol. Theprecipitated RNA was recovered by centrifugation at 12,000×g for 30minutes at 4° C. The final pellet was washed with cold 70% ethanol, airdried, and resuspended in 500 ml of diethylpyrocarbonate treated water(DEPC-water).

The quality and quantity of the purified RNA was assessed with anAGILENT® 2100 Bioanalyzer (Agilent Technologies, Inc., Palo Alto,Calif., USA). Polyadenylated mRNA was isolated from 360 μg of total RNAwith the aid of a POLY(A)PURIST™ Magnetic Kit (Ambion, Inc., Austin,Tex., USA) according to the manufacturer's instructions.

To create a cDNA library, a CLONEMINE® Kit (Invitrogen Corp., Carlsbad,Calif., USA) was employed to construct a directional library that doesnot require the use of restriction enzyme cloning, thereby reducing thenumber of chimeric clones and size bias.

To insure the successful synthesis of the cDNA, two reactions wereperformed in parallel with two different concentrations of mRNA (2.2 and4.4 μg of poly (A)⁺ mRNA). The mRNA samples were mixed with aBiotin-attB2-Oligo(dt) primer (Invitrogen Corp., Carlsbad, Calif., USA),1× first strand buffer (Invitrogen Corp., Carlsbad, Calif., USA), 2 μlof 0.1 M dithiothreitol (DTT), 10 mM of each dNTP, and water to a finalvolume of 18 and 16 μl, respectively.

The reaction mixtures were mixed and then 2 and 4 μl of SUPERSCRIPT™reverse transcriptase (Invitrogen Corp., Carlsbad, Calif., USA),respectively, were added. The reaction mixtures were incubated at 45° C.for 60 minutes to synthesize the first complementary strand. For secondstrand synthesis, to each first strand reaction was added 30 μl of 5×second strand buffer (Invitrogen Corp., Carlsbad, Calif., USA), 3 μl of10 mM of each dNTP, 10 units of E. coli DNA ligase (Invitrogen Corp.,Carlsbad, Calif., USA), 40 units of E. coli DNA polymerase I (InvitrogenCorp., Carlsbad, Calif., USA), and 2 units of E. coli RNase H(Invitrogen Corp., Carlsbad, Calif., USA) in a total volume of 150 μl.The mixtures were then incubated at 16° C. for two hours. After thetwo-hour incubation 2 μl of T4 DNA polymerase (Invitrogen Corp.,Carlsbad, Calif., USA) were added to each reaction and incubated at 16°C. for 5 minutes to create a bunt-ended cDNA. The cDNA reactions wereextracted with a mixture of phenol-chloroform-isoamyl alcohol 25:24:1v/v/v and precipitated in the presence of 20 μg of glycogen, 120 μl of 5M ammonium acetate, and 660 μl of ethanol. After centrifugation at12,000×g for 30 minutes at 4° C., the cDNA pellets were washed with cold70% ethanol, dried under vacuum for 2-3 minutes, and resuspended in 18μl of DEPC-water. To each resuspended cDNA sample were added 10 μl of 5×adapted buffer (Invitrogen, Carlsbad, Calif.), 10 μg of each attB1adapter (Invitrogen, Carlsbad, Calif., USA), 7 μl of 0.1 M DTT, and 5units of T4 DNA ligase (Invitrogen, Carlsbad, Calif., USA). Ligationreactions were incubated overnight at 16° C. Excess adapters wereremoved by size-exclusion chromatography in 1 ml of SEPHACRYL™ S-500 HRresin (Amersham Biosciences, Piscataway, N.J., USA). Column fractionswere collected according to the CLONEMINE® Kit's instructions andfractions 3 to 14 were analyzed with an AGILENT® 2100 Bioanalyzer todetermine the fraction at which the attB1 adapters started to elute.This analysis showed that the adapters started eluting around fraction10 or 11. For the first library fractions 6 to 11 were pooled and forthe second library fractions 4-11 were pooled.

Cloning of the cDNA was performed by homologous DNA recombinationaccording to the GATEWAY® System protocol (Invitrogen Corp., Carlsbad,Calif., USA) using BP CLONASE™ (Invitrogen Corp., Carlsbad, Calif., USA)as the recombinase. Each BP CLONASE™ recombination reaction containedapproximately 70 ng of attB-flanked-cDNA, 250 ng of pDONR™ 222, 2 μl of5×BP CLONASE™ buffer, 2 μl of TE, and 3 μl of BP CLONASE™. All reagentswere obtained from Invitrogen, Carlsbad, Calif., USA. Recombinationreactions were incubated at 25° C. overnight.

Heat-inactivated BP recombination reactions were then divided into 6aliquots and electroporated into ELECTROMAX™ DH10B electrocompetentcells (Invitrogen Corp., Carlsbad, Calif., USA) using a GENE PULSE®(Bio-Rad Laboratories, Inc., Hercules, Calif., USA) with the followingparameters: Voltage: 2.0 kV; Resistance: 200Ω; and Capacity: 25 μF.Electroporated cells were resuspended in 1 ml of SOC medium andincubated at 37° C. for 60 minutes with constant shaking at 200 rpm.After the incubation period, the transformed cells were pooled and mixed1:1 with freezing medium. A 200 μl aliquot was removed from each libraryfor library titration and then the rest of each library was aliquotedinto 1.8 ml cryovials (Wheaton Science Products, Millville, N.J., USA)and stored frozen at −80° C.

Four serial dilutions of each library were prepared: 1/100, 1/1000,1/10⁴, and 1/10⁵. From each dilution, 100 μl were plated onto 150 mm LBplates supplemented with 50 μg of kanamycin per ml and incubated at 37°C. overnight. The number of colonies on each dilution plate was countedand used to calculate the total number of transformants in each library.

The first library contained approximately 5.4 million independent clonesand the second library contained approximately 9 million independentclones.

Example 2 Template Preparation and Nucleotide Sequencing of cDNA Clones

Aliquots from both libraries described in Example 1 were mixed andplated onto 25×25 cm LB plates supplemented with 50 μg of kanamycin perml. Individual colonies were arrayed onto 96-well plates containing 100μl of LB medium supplemented with 50 μg of kanamycin per ml with the aidof a QPix Robot (Genetix Inc., Boston, Mass., USA). Forty-five 96-wellplates were obtained for a total of 4320 individual clones. The plateswere incubated overnight at 37° C. with shaking at 200 rpm. Afterincubation, 100 μl of sterile 50% glycerol was added to each well. Thetransformants were replicated with the aid of a 96-pin tool (Boekel,Feasterville, Pa., USA) into secondary, deep-dish 96-well microcultureplates (Advanced Genetic Technologies Corporation, Gaithersburg, Md.,USA) containing 1 ml of MAGNIFICENT BROTH™ (MacConnell Research, SanDiego, Calif., USA) supplemented with 50 μg of kanamycin per ml in eachwell. The primary microtiter plates were stored frozen at −80° C. Thesecondary deep-dish plates were incubated at 37° C. overnight withvigorous agitation at 300 rpm on a rotary shaker. To prevent spillingand cross-contamination, and to allow sufficient aeration, eachsecondary culture plate was covered with a polypropylene pad (AdvancedGenetic Technologies Corporation, Gaithersburg, Md., USA) and a plasticmicrotiter dish cover. Plasmid DNA was prepared with a Robot-Smart 384(MWG Biotech Inc., High Point, N.C., USA) and a MONTAGE™ PlasmidMiniprep Kit (Millipore, Billerica, Mass., USA).

Sequencing reactions were performed using BIGDYE® (Applied Biosystems,Inc., Foster City, Calif., USA) terminator chemistry (Giesecke et al.,1992, Journal of Virology Methods 38: 47-60) and a M13 Forward (−20)sequencing primer: 5′-GTAAAACGACGGCCAG-3′ (SEQ ID NO: 3)

The sequencing reactions were performed in a 384-well format with aRobot-Smart 384. Terminator removal was performed with a MULTISCREEN®Seq384 Sequencing Clean-up Kit (Millipore, Billerica, Mass., USA).Reactions contained 6 μl of plasmid DNA and 4 μl of sequencingmaster-mix (Applied Biosystems, Foster City, Calif., USA) containing 1μl of 5× sequencing buffer (Millipore, Billerica, Mass., USA), 1 μl ofBIGDYE® terminator (Applied Biosystems, Inc., Foster City, Calif., USA),1.6 pmoles of M13 Forward primer, and 1 μl of water. Single-pass DNAsequencing was performed with an ABI PRISM Automated DNA Sequencer Model3700 (Applied Biosystems, Foster City, Calif., USA).

Example 3 Analysis of DNA Sequence Data of cDNA Clones

Base calling, quality value assignment, and vector trimming wereperformed with the assistance of PHRED/PHRAP software (University ofWashington, Seattle, Wash., USA). Clustering analysis of the ESTs wasperformed with a Transcript Assembler v. 2.6.2. (Paracel, Inc.,Pasadena, Calif., USA). Analysis of the EST clustering indicated thepresence of 395 independent clusters.

Sequence homology analysis of the assembled EST sequences againstdatabases was performed with the Blastx program (Altschul et. al., 1990,J. Mol. Biol. 215:403-410) on a 32-node Linux cluster (Paracel, Inc.,Pasadena, Calif., USA) using the BLOSUM 62 matrix (Henikoff, 1992, Proc.Natl. Acad. Sci. USA 89: 10915-10919).

Example 4 Identification of cDNA Clones Encoding a Thielavia terrestrisPolypeptide Having Aspartic Endopeptidase Activity

A cDNA clone encoding a Thielavia terrestris polypeptide having asparticendopeptidase activity was initially identified by sequence homology toa characterized endothiapepsin from Cryphonectria parasitica(Razanamparany, et al., 1993, Gene 125:135-141, UniProt accession numberP11838.

After this initial identification, the clone, designated Tter30A5, wasretrieved from the original frozen stock plate and streaked onto a LBplate supplemented with 50 μg of kanamycin per ml. The plate wasincubated overnight at 37° C. and the next day a single colony from theplate was used to inoculate 3 ml of LB medium supplemented with 150 μgof kanamycin per ml. The liquid culture was incubated overnight at 37°C. and plasmid DNA was prepared with a BIOROBOT® 9600 (QIAGEN, Inc.,Valencia, Calif., USA). Using a primer walking strategy, the insertedcDNA in the Tter30A5 plasmid was completely sequenced.

Analysis of the deduced protein sequence of Tter30A5 with theInterproscan program (Zdobnov and Apweiler, 2001, Bioinformatics 17:847-8) showed that the gene encoded by Tter30A5 contained the aspartylprotease sequence signature known as the PF00026. This sequencesignature is located at amino acids position 135 through 465 in thededuced peptide sequence (SEQ ID NO: 2).

The cDNA sequence (SEQ ID NO: 1) and deduced amino acid sequence (SEQ IDNO: 2) of the Thielavia terrestris aspartic endopeptidase gene are shownin FIG. 1. The cDNA clone encodes a polypeptide of 467 amino acids. The% G+C content of the coding sequence of the gene is 65.0% and of themature protein coding region (nucleotides 61 to 1401 of SEQ ID NO: 1) is65.8%. Using the SignalP software program (Nielsen et al., 1997, ProteinEngineering 10:1-6), a signal peptide of 20 residues was predicted. Thepredicted mature protein contains 447 amino acids with a molecular massof 47.3 kDa.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program ofEMBOSS with a gap open penalty of 10, gap extension penalty of 0.5, andthe EBLOSUM62 matrix. The alignment showed that the deduced amino acidsequence of the Thielavia terrestris polypeptide having asparticendopeptidase activity gene shared 60% identity (excluding gaps) to thededuced amino acid sequences of putative uncharacterized protein(Uniprot accession number Q2HFJ8) from Chaetomium globosum and 44%identity to a characterized aspartic proteinase from Glomerellacingulata (Clark et al., 1997, Microbiology 143 (Pt 4):1395-403),UniProt accession number Q00895.

Once the identity of Tter30A5 was confirmed, a 0.5 μl aliquot of plasmidDNA from this clone (pTter30A5, FIG. 2) was transferred into a vial ofE. coli TOP10 cells (Invitrogen Corp., Carlsbad, Calif., USA), gentlymixed, and incubated on ice for 10 minutes. The cells were thenheat-shocked at 42° C. for 30 seconds and incubated again on ice for 2minutes. The cells were resuspended in 250 μl of SOC medium andincubated at 37° C. for 60 minutes with constant shaking at 200 rpm.After the incubation period, two 30 μl aliquots were plated onto LBplates supplemented with 50 μg of kanamycin per ml and incubatedovernight at 37° C. The next day a single colony was picked and streakedonto three 1.8 ml cryovials containing about 1.5 ml of LB agarosesupplemented with 50 μg of kanamycin per ml. The vials were sealed withPETRISEAL™ (Diversified Biotech, Boston Mass., USA) and deposited withthe Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center, Peoria, Ill., USA, as NRRL B-50209, with adeposit date of Dec. 11, 2008.

DEPOSIT OF BIOLOGICAL MATERIAL

The following biological material has been deposited under the terms ofthe Budapest Treaty with the Agricultural Research Service PatentCulture Collection (NRRL), Northern Regional Research Center, 1815University Street, Peoria, Ill., USA, and given the following accessionnumber:

Deposit Accession Number Date of Deposit E. coli pTter30A5 NRRL B-50209Dec. 11, 2008

The strain has been deposited under conditions that assure that accessto the culture will be available during the pendency of this patentapplication to one determined by foreign patent laws to be entitledthereto. The deposit represents a substantially pure culture of thedeposited strain. The deposit is available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

The present invention is further described by the following paragraphs:

[1] An isolated polypeptide having aspartic endopeptidase activity,selected from the group consisting of: (a) a polypeptide comprising anamino acid sequence having at least 60% sequence identity to the maturepolypeptide of SEQ ID NO: 2; (b) a polypeptide encoded by apolynucleotide that hybridizes under at least medium stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the genomic DNA sequence comprising the mature polypeptidecoding sequence of SEQ ID NO: 1, or (iii) a full-length complementarystrand of (i) or (ii); (c) a polypeptide encoded by a polynucleotidecomprising a nucleotide sequence having at least 60% sequence identityto the mature polypeptide coding sequence of SEQ ID NO: 1; and (d) avariant comprising a substitution, deletion, and/or insertion of one ormore (several) amino acids of the mature polypeptide of SEQ ID NO: 2.

[2] The polypeptide of paragraph 1, comprising an amino acid sequencehaving at least 60% sequence identity to the mature polypeptide of SEQID NO: 2.

[3] The polypeptide of paragraph 2, comprising an amino acid sequencehaving at least 65% sequence identity to the mature polypeptide of SEQID NO: 2.

[4] The polypeptide of paragraph 3, comprising an amino acid sequencehaving at least 70% sequence identity to the mature polypeptide of SEQID NO: 2.

[5] The polypeptide of paragraph 4, comprising an amino acid sequencehaving at least 75% sequence identity to the mature polypeptide of SEQID NO: 2.

[6] The polypeptide of paragraph 5, comprising an amino acid sequencehaving at least 80% sequence identity to the mature polypeptide of SEQID NO: 2.

[7] The polypeptide of paragraph 6, comprising an amino acid sequencehaving at least 85% sequence identity to the mature polypeptide of SEQID NO: 2.

[8] The polypeptide of paragraph 7, comprising an amino acid sequencehaving at least 90% sequence identity to the mature polypeptide of SEQID NO: 2.

[9] The polypeptide of paragraph 8, comprising an amino acid sequencehaving at least 95% sequence identity to the mature polypeptide of SEQID NO: 2.

[10] The polypeptide of paragraph 9, comprising an amino acid sequencehaving at least 97% sequence identity to the mature polypeptide of SEQID NO: 2.

[11] The polypeptide of paragraph 1, comprising or consisting of theamino acid sequence of SEQ ID NO: 2; or a fragment thereof havingaspartic endopeptidase activity.

[12] The polypeptide of paragraph 11, comprising or consisting of theamino acid sequence of SEQ ID NO: 2.

[13] The polypeptide of paragraph 1, comprising or consisting of themature polypeptide of SEQ ID NO: 2.

[14] The polypeptide of paragraph 1, which is encoded by apolynucleotide that hybridizes under at least medium stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the genomic DNA sequence comprising the mature polypeptidecoding sequence of SEQ ID NO: 1, or (iii) a full-length complementarystrand of (i) or (ii).

[15] The polypeptide of paragraph 14, which is encoded by apolynucleotide that hybridizes under at least medium-high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the genomic DNA sequence comprising the mature polypeptidecoding sequence of SEQ ID NO: 1, or (iii) a full-length complementarystrand of (i) or (ii).

[16] The polypeptide of paragraph 15, which is encoded by apolynucleotide that hybridizes under at least high stringency conditionswith (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii)the genomic DNA sequence comprising the mature polypeptide codingsequence of SEQ ID NO: 1, or (iii) a full-length complementary strand of(i) or (ii).

[17] The polypeptide of paragraph 16, which is encoded by apolynucleotide that hybridizes under at least very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the genomic DNA sequence comprising the mature polypeptidecoding sequence of SEQ ID NO: 1, or (iii) a full-length complementarystrand of (i) or (ii).

[18] The polypeptide of paragraph 1, which is encoded by apolynucleotide comprising a nucleotide sequence having at least 60%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1.

[19] The polypeptide of paragraph 18, which is encoded by apolynucleotide comprising a nucleotide sequence having at least 65%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1.

[20] The polypeptide of paragraph 19, which is encoded by apolynucleotide comprising a nucleotide sequence having at least 70%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1.

[21] The polypeptide of paragraph 20, which is encoded by apolynucleotide comprising a nucleotide sequence having at least 75%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1.

[22] The polypeptide of paragraph 21, which is encoded by apolynucleotide comprising a nucleotide sequence having at least 80%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1.

[23] The polypeptide of paragraph 22, which is encoded by apolynucleotide comprising a nucleotide sequence having at least 85%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1.

[24] The polypeptide of paragraph 23, which is encoded by apolynucleotide comprising a nucleotide sequence having at least 90%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1.

[25] The polypeptide of paragraph 24, which is encoded by apolynucleotide comprising a nucleotide sequence having at least 95%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1.

[26] The polypeptide of paragraph 25, which is encoded by apolynucleotide comprising a nucleotide sequence having at least 97%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1.

[27] The polypeptide of paragraph 1, which is encoded by apolynucleotide comprising or consisting of the nucleotide sequence ofSEQ ID NO: 1; or a subsequence thereof encoding a fragment havingaspartic endopeptidase activity.

[28] The polypeptide of paragraph 27, which is encoded by apolynucleotide comprising or consisting of the nucleotide sequence ofSEQ ID NO: 1.

[29] The polypeptide of paragraph 1, which is encoded by apolynucleotide comprising or consisting of the mature polypeptide codingsequence of SEQ ID NO: 1.

[30] The polypeptide of paragraph 1, wherein the polypeptide is avariant comprising a substitution, deletion, and/or insertion of one ormore (several) amino acids of the mature polypeptide of SEQ ID NO: 2.

[31] The polypeptide of paragraph 1, which is encoded by thepolynucleotide contained in plasmid pTter30A5 which is contained in E.coli NRRL B-50209.

[32] The polypeptide of any of paragraphs 1-31, wherein the maturepolypeptide is amino acids 21 to 467 of SEQ ID NO: 2.

[33] The polypeptide of any of paragraphs 1-32, wherein the maturepolypeptide coding sequence is nucleotides 61 to 1401 of SEQ ID NO: 1.

[34] An isolated polynucleotide comprising a nucleotide sequence thatencodes the polypeptide of any of paragraphs 1-33.

[35] The isolated polynucleotide of paragraph 34, comprising at leastone mutation in the mature polypeptide coding sequence of SEQ ID NO: 1,in which the mutant nucleotide sequence encodes the mature polypeptideof SEQ ID NO: 2.

[36] A nucleic acid construct comprising the polynucleotide of paragraph34 or 35 operably linked to one or more (several) control sequences thatdirect the production of the polypeptide in an expression host.

[37] A recombinant expression vector comprising the polynucleotide ofparagraph 34 or 35.

[38] A recombinant host cell comprising the polynucleotide of paragraph34 or 35 operably linked to one or more (several) control sequences thatdirect the production of a polypeptide having aspartic endopeptidaseactivity.

[39] A method of producing the polypeptide of any of paragraphs 1-33,comprising: (a) cultivating a cell, which in its wild-type form producesthe polypeptide, under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide.

[40] A method of producing the polypeptide of any of paragraphs 1-33,comprising: (a) cultivating a host cell comprising a nucleic acidconstruct comprising a nucleotide sequence encoding the polypeptideunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

[41] A method of producing a mutant of a parent cell, comprisingdisrupting or deleting a polynucleotide encoding the polypeptide, or aportion thereof, of any of paragraphs 1-33, which results in the mutantproducing less of the polypeptide than the parent cell.

[42] A mutant cell produced by the method of paragraph 41.

[43] The mutant cell of paragraph 42, further comprising a gene encodinga native or heterologous protein.

[44] A method of producing a protein, comprising: (a) cultivating themutant cell of paragraph 43 under conditions conducive for production ofthe protein; and (b) recovering the protein.

[45] The isolated polynucleotide of paragraph 34 or 35, obtained by (a)hybridizing a population of DNA under at least medium stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the genomic DNA sequence comprising the mature polypeptidecoding sequence of SEQ ID NO: 1, or (iii) a full-length complementarystrand of (i) or (ii); and (b) isolating the hybridizing polynucleotide,which encodes a polypeptide having aspartic endopeptidase activity.

[46] The isolated polynucleotide of paragraph 45, obtained by (a)hybridizing a population of DNA under at least medium-high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the genomic DNA sequence comprising the mature polypeptidecoding sequence of SEQ ID NO: 1, or (iii) a full-length complementarystrand of (i) or (ii); and (b) isolating the hybridizing polynucleotide,which encodes a polypeptide having aspartic endopeptidase activity.

[47] The isolated polynucleotide of paragraph 46, obtained by (a)hybridizing a population of DNA under at least high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the genomic DNA sequence comprising the mature polypeptidecoding sequence of SEQ ID NO: 1, or (iii) a full-length complementarystrand of (i) or (ii); and (b) isolating the hybridizing polynucleotide,which encodes a polypeptide having aspartic endopeptidase activity.

[48] The isolated polynucleotide of paragraph 47, obtained by (a)hybridizing a population of DNA under at least very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the genomic DNA sequence comprising the mature polypeptidecoding sequence of SEQ ID NO: 1, or (iii) a full-length complementarystrand of (i) or (ii); and (b) isolating the hybridizing polynucleotide,which encodes a polypeptide having aspartic endopeptidase activity.

[49] The isolated polynucleotide of any of paragraphs 45-48, wherein themature polypeptide coding sequence is nucleotides 61 to 1401 of SEQ IDNO: 1.

[50] A method of producing a polynucleotide comprising a mutantnucleotide sequence encoding a polypeptide having aspartic endopeptidaseactivity, comprising: (a) introducing at least one mutation into themature polypeptide coding sequence of SEQ ID NO: 1, wherein the mutantnucleotide sequence encodes a polypeptide comprising or consisting ofthe mature polypeptide of SEQ ID NO: 2; and (b) recovering thepolynucleotide comprising the mutant nucleotide sequence.

[51] A mutant polynucleotide produced by the method of paragraph 50.

[52] A method of producing a polypeptide, comprising: (a) cultivating acell comprising the mutant polynucleotide of paragraph 51 encoding thepolypeptide under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide.

[53] A method of producing the polypeptide of any of paragraphs 1-33,comprising: (a) cultivating a transgenic plant or a plant cellcomprising a polynucleotide encoding the polypeptide under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide.

[54] A transgenic plant, plant part or plant cell transformed with apolynucleotide encoding the polypeptide of any of paragraphs 1-33.

[55] A double-stranded inhibitory RNA (dsRNA) molecule comprising asubsequence of the polynucleotide of paragraph 34 or 35, whereinoptionally the dsRNA is a siRNA or a miRNA molecule.

[56] The double-stranded inhibitory RNA (dsRNA) molecule of paragraph55, which is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or moreduplex nucleotides in length.

[57] A method of inhibiting the expression of a polypeptide havingaspartic endopeptidase activity in a cell, comprising administering tothe cell or expressing in the cell a double-stranded RNA (dsRNA)molecule, wherein the dsRNA comprises a subsequence of thepolynucleotide of paragraph 34 or 35.

[58] The method of paragraph 57, wherein the dsRNA is about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in length.

[59] An isolated polynucleotide encoding a signal peptide comprising orconsisting of amino acids 1 to 20 of SEQ ID NO: 2.

[60] A nucleic acid construct comprising a gene encoding a proteinoperably linked to the polynucleotide of paragraph 59, wherein the geneis foreign to the polynucleotide encoding the signal peptide.

[61] A recombinant expression vector comprising a gene encoding aprotein operably linked to the polynucleotide of paragraph 59, whereinthe gene is foreign to the polynucleotide encoding the signal peptide.

[62] A recombinant host cell comprising a gene encoding a proteinoperably linked to the polynucleotide of paragraph 59, wherein the geneis foreign to the polynucleotide encoding the signal peptide.

[63] A method of producing a protein, comprising: (a) cultivating arecombinant host cell comprising a gene encoding a protein operablylinked to the polynucleotide of paragraph 59, wherein the gene isforeign to the polynucleotide encoding the signal peptide, underconditions conducive for production of the protein; and (b) recoveringthe protein.

[64] A composition comprising the polypeptide of any of paragraphs 1-33.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

What is claimed is:
 1. A nucleic acid construct comprising an isolatedpolynucleotide comprising a nucleotide sequence that encodes apolypeptide having aspartic endopeptidase activity, wherein thepolynucleotide is operably linked to one or more heterologous controlsequences that direct the production of the polypeptide in a recombinanthost cell, and wherein the polypeptide having aspartic endopeptidaseactivity is selected from the group consisting of: (a) a polypeptidecomprising an amino acid sequence having at least 95% sequence identityto the mature polypeptide of SEQ ID NO: 2; (b) a polypeptide encoded bya polynucleotide comprising a nucleotide sequence having at least 95%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1; (c) a polypeptide comprising the amino acid sequence of SEQ IDNO: 2 or the mature polypeptide thereof; or a fragment thereof havingaspartic endopeptidase activity of at least 380 amino acids; and (d) apolypeptide encoded by a polynucleotide comprising the nucleotidesequence of SEQ ID NO: 1 or the mature polypeptide coding sequencethereof.
 2. An isolated recombinant host cell comprising the nucleicacid construct of claim
 1. 3. A method of producing a polypeptide havingaspartic endopeptidase activity, comprising: (a) cultivating therecombinant host cell of claim 2 under conditions conducive forproduction of the polypeptide; and (b) recovering the polypeptide.
 4. Amethod of producing a polypeptide having aspartic endopeptidaseactivity, comprising: (a) cultivating a transgenic plant or a plant cellcomprising the nucleic acid construct of claim 1 under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide.
 5. A transgenic plant, plant part or plant cell transformedwith the nucleic acid construct of claim
 1. 6. A nucleic acid constructcomprising a gene encoding a protein operably linked to a polynucleotideencoding a signal peptide comprising amino acids 1 to 20 of SEQ ID NO:2, wherein the gene is foreign to the polynucleotide encoding the signalpeptide.
 7. A method of producing a protein, comprising: (a) cultivatinga recombinant host cell comprising the nucleic acid construct of claim 6under conditions conducive for production of the protein; and (b)recovering the protein.
 8. The nucleic acid construct of claim 1,wherein the polypeptide comprises an amino acid sequence having at least96% sequence identity to the mature polypeptide of SEQ ID NO:
 2. 9. Thenucleic acid construct of claim 1, wherein the polypeptide comprises anamino acid sequence having at least 97% sequence identity to the maturepolypeptide of SEQ ID NO:
 2. 10. The nucleic acid construct of claim 1,wherein the polypeptide comprises an amino acid sequence having at least98% sequence identity to the mature polypeptide of SEQ ID NO:
 2. 11. Thenucleic acid construct of claim 1, wherein the polypeptide comprises anamino acid sequence having at least 99% sequence identity to the maturepolypeptide of SEQ ID NO:
 2. 12. The nucleic acid construct of claim 1,wherein the polypeptide comprises the mature polypeptide of SEQ ID NO:2.
 13. The nucleic acid construct of claim 12, wherein the maturepolypeptide is amino acids 21 to 467 of SEQ ID NO:
 2. 14. The nucleicacid construct of claim 1, wherein the polypeptide is encoded by apolynucleotide comprising a nucleotide sequence having at least 96%sequence identity to the mature polypeptide coding sequence of SEQ IDNO:
 1. 15. The nucleic acid construct of claim 1, wherein thepolypeptide is encoded by a polynucleotide comprising a nucleotidesequence having at least 97% sequence identity to the mature polypeptidecoding sequence of SEQ ID NO:
 1. 16. The nucleic acid construct of claim1, wherein the polypeptide is encoded by a polynucleotide comprising anucleotide sequence having at least 98% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO:
 1. 17. The nucleic acidconstruct of claim 1, wherein the polypeptide is encoded by apolynucleotide comprising a nucleotide sequence having at least 99%sequence identity to the mature polypeptide coding sequence of SEQ IDNO:
 1. 18. The nucleic acid construct of claim 1, wherein thepolypeptide is encoded by a polynucleotide comprising the maturepolypeptide coding sequence of SEQ ID NO:
 1. 19. The nucleic acidconstruct of claim 18, wherein the mature polypeptide coding sequence isnucleotides 61 to 1401 of SEQ ID NO:
 1. 20. The nucleic acid constructof claim 1, wherein the polypeptide is encoded by the polynucleotidecontained in plasmid pTter30A5 which is contained in E. coli NRRLB-50209.
 21. The nucleic acid construct of claim 1, wherein thepolypeptide comprises an amino acid sequence having at least 95%sequence identity to the mature polypeptide of SEQ ID NO:
 2. 22. Anisolated recombinant host cell transformed with a nucleic acid constructcomprising a polynucleotide comprising a nucleotide sequence thatencodes a polypeptide having aspartic endopeptidase activity, whereinthe polynucleotide is operably linked to one or more control sequencesthat direct the production of the polypeptide in the recombinant hostcell, wherein said nucleotide sequence is heterologous to therecombinant host cell, and wherein the polypeptide having asparticendopeptidase activity is selected from the group consisting of: (a) apolypeptide comprising an amino acid sequence having at least 95%sequence identity to the mature polypeptide of SEQ ID NO: 2; (b) apolypeptide encoded by a polynucleotide comprising a nucleotide sequencehaving at least 95% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 1; (c) a polypeptide comprising the amino acidsequence of SEQ ID NO: 2 or the mature polypeptide thereof; or afragment thereof having aspartic endopeptidase activity of at least 380amino acids; and (d) a polypeptide encoded by a polynucleotidecomprising the nucleotide sequence of SEQ ID NO: 1 or the maturepolypeptide coding sequence thereof.
 23. The recombinant host cell ofclaim 22, wherein the polypeptide comprises an amino acid sequencehaving at least 95% sequence identity to the mature polypeptide of SEQID NO:
 2. 24. The recombinant host cell of claim 22, wherein thepolypeptide comprises an amino acid sequence having at least 96%sequence identity to the mature polypeptide of SEQ ID NO:
 2. 25. Therecombinant host cell of claim 22, wherein the polypeptide comprises anamino acid sequence having at least 97% sequence identity to the maturepolypeptide of SEQ ID NO:
 2. 26. The recombinant host cell of claim 22,wherein the polypeptide comprises an amino acid sequence having at least98% sequence identity to the mature polypeptide of SEQ ID NO:
 2. 27. Therecombinant host cell of claim 22, wherein the polypeptide comprises anamino acid sequence having at least 99% sequence identity to the maturepolypeptide of SEQ ID NO:
 2. 28. The recombinant host cell of claim 22,wherein the polypeptide comprises the mature polypeptide of SEQ ID NO:2.
 29. The recombinant host cell of claim 22, wherein the maturepolypeptide is amino acids 21 to 467 of SEQ ID NO:
 2. 30. A method ofproducing a polypeptide having aspartic endopeptidase activity,comprising: (a) cultivating the recombinant host cell of claim 22 underconditions conducive for production of the polypeptide; and (b)recovering the polypeptide.